This invention is directed to an instrument for making chemical and biological analyses of a sample using surface plasmon resonance. Surface plasmon resonance (SPR) is a method whereby surface plasmons or surface electromagnetic waves, that are characteristic of conducting metal surfaces, are propagated parallel to a metal/dielectric interface. This phenomenon is accomplished in the IR-visible wavelength region for air/metal and water/metal interfaces. In one simple form, SPR reflectivity measurements can be used to detect and identify unknown molecules such as DNA or proteins by the changes in the local index of refraction upon adsorption of the target molecule to the metal surface.
Surface plasmon resonance (SPR) is used in the nondestructive study of surfaces, interfaces, and very thin layers, and has recently been found to be particularly adapted for the study of immunologic phenomenon such as antigen-antibody reactions and protein-protein interactions. A surface plasmon is an oscillation of free electrons propagated along the surface of a conductor which is typically in the form of a thin metal film of gold, silver or copper. Transverse-magnetic (TM) polarized energy in an evanescent field excites surface plasmons on the thin metal film. The characteristics of the resonance are directly related to the refractive indices of materials on both sides of the metal film. By including the sample to be measured as a layer on one side of the metal film, changes in the refractive index of the sample can be monitored by measuring changes in the surface plasmon resonance response. Measurement of the SPR response is done by monitoring the characteristics of reflected light at incident angles above the critical angle.
The surface selectivity of SPR arises from the enhancement of the optical electric fields at metal surfaces when surface plasmon polaritons (SPPs) are created at the metal/dielectric surface. SPPs are coupled photon-plasmon surface electromagnetic waves that propagate parallel to the metal/dielectric interface. The intensity of the optical electric fields associated with an SPP decays exponentially in distance away from the metal surface, with a typical decay length for an SPP into the dielectric being on the order of 200 nm. SPPs cannot be created on an isolated planar metal surface, but rather require a prism or grating coupling geometry for exciting SPPs. Thus, surface plasmon resonance is achieved by using the evanescent wave which is generated when a p-polarized light beam is totally internally reflected at the boundary of a medium having a high dielectric constant, such as glass. The free electron oscillation is affected by the refractive index of the material adjacent the metal surface which forms the basis of SPR measurements.
In a typical SPR scanning angle experiment, p-polarized light from a laser is directed through a prism onto a metal film on which is disposed a thin sample layer being studied. The prism-sample assembly is mounted to a rotation stage, which allows scanning of the incident angle of the laser beam. As the angle of incidence of the laser beam is varied, surface plasmon resonance is evidenced as a sharp dip in the intensity of the laser beam internally reflected within the prism at a particular angle of incidence. The angle of incidence at which resonance occurs is affected by the refractive index of the thin sample layer disposed on the metal film. The angle of incidence corresponding to resonance is thus a direct measure of the characteristics of the thin sample layer. In the case of immunoassays, the measured angle of incidence corresponding to resonance represents a direct measure of the state of reaction between an antibody and its antigen.
A typical SPR instrument employs a method of varying the incident light angle and detecting the internally reflected light by using a θ-2θ mechanical stage. The classical θ-2θ mechanical stage has a fixed input light source, a rotating optical element with θ motion, and a tracking detector stage with 2θ motion. In the prism SPR configuration, as the angle of incidence is varied by rotating the prism optical element x degrees, the detector must rotate 2x degrees to track the beam. Many SPR systems are constructed using this method. FIG. 1 shows the typical layout of such a system.
While the information determined using typical SPR is immense, there are several disadvantages of a system with this type of θ-2θ stage. Such disadvantages include: the SPR sensor chip is in a vertical or other non-horizontal format making experimental access more difficult; the prism and SPR chip optical elements are on the rotating platform resulting in a non-rigid mount, causing optical instability; the heater control is more difficult to implement because of a rotating non-rigid platform; tracking detector is on a pivot arm that is a moveable non-rigid structure; the detector and detector optics mass require significant torque to rotate; and a gearbox or synchronous rotating stages are required, with resultant mechanical backlash.
Codner et al. described a horizontal SPR instrument in U.S. patent application Ser. No. 10/602,243. While Codner et al. describe a horizontal SPR apparatus that provides for a horizontal SPR surface orientation, this apparatus requires a complex coordination of upper and lower mirrors in relation to the prism and the sample. In order to accomplish this task, the device taught by Codner requires a four-bar linkage system to maintain the beam path as the light is scanned along the sample surface. This design provides a fixed and horizontal SPR chip orientation, however, with a four-mirror system with complex and interacting degrees of freedom resulting in a mechanically complex apparatus.
Similarly, Codner et al. describe a portable SPR instrument in U.S. patent application Ser. No. 10/411,583 (the '583 application) that is designed to provide a device that is smaller, easily transported and can be used outside of the laboratory. However, in order to provide the smaller footprint and portability the device described in the '583 application requires the light source and imaging detector be supported on linked swing arms, limiting the angular range of travel and restricting the type and size of components utilized in the light source and detector assemblies.
Due to these deficits, it would be desirable to have a surface plasmon resonance apparatus that has a fixed input light source, a fixed prism element with horizontally orientated SPR chip attached, with a fixed detector stage allowing for a more compact and less complex apparatus that still provides the versatility of light source and detector systems as larger more complex models.